The outcome of a viral infection is determined by the agent's
pathogenicity and by host factors, such as genetic predisposition. For
RNA viruses, which are notorious for their swift evolution and
adaptation, a pathogen's specific genotype usually is to blame for
devastating effects (1). In many cases, however, the virus genome is not
easy to search for particular mutations because recovery of complete
viral sequences from clinical specimens remains extremely difficult.
This is especially true for hantaviruses (family Bunyaviridae) that
cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus
cardiopulmonary syndrome (2,3). Thus far, only a few complete hantavirus
genomes originating from persons with clinical cases have been reported
(4,5), and only 1 was recovered without passaging first in cell culture
(4), which by itself can induce adaptive changes in the viral genome
(6). We present the complete genome of PUUV directly recovered from a
person with fatal infection.

Usually PUUV causes mild HFRS (also called nephropathia epidemica
[NE]). In Finland, 1,000-3,000 NE cases are diagnosed annually, i.e.,
[approximately equals] 60 cases/100,000 persons during years when the
vole population peaks (7). Almost 100% of infected persons recover, and
long-lasting complications are rare. The few fatal cases reported (8,9)
showed no apparent geographic clustering. Thus, whether more severe
illness could be connected to certain genetic variants of PUUV remains
unknown.

The Study

The patient was a previously healthy 37-year-old man with a history
of smoking. He died in November 2008 of severe NE on day 4 after the
onset of symptoms that started with high fever, vomiting and diarrhea,
headache, and visual disturbances. His condition deteriorated quickly,
and multiorgan failure developed, including respiratory distress, acute
kidney failure, liver failure, and severe thrombocytopenia.

A standard autopsy was performed, and tissue samples were stored
fresh at -70[degrees]C and fixed in formalin. PUUV infection was
confirmed initially by IgM test and later by reverse transcription PCR
(RT-PCR), followed by sequencing. Genetic analysis was performed from
autopsy samples stored fresh at -70[degrees]C (the high quality of
clinical samples was crucial for the downstream applications). Complete
PUUV genomes were recovered in a set of nested and seminested PCR
(sequencers of primers are available on request). Amplicons were
gel-purified and sequenced directly by using ABI PRISM Dye Terminator
sequencing kit (PerkinElmer/ABI, Foster City, CA, USA). Quantitative
RT-PCR was used to measure PUUV load with DyNAmo Capillary SYBR Green
kit (Finnzymes, Espoo, Finland). Copy numbers were calculated from a
standard curve created by using in vitro transcribed PUUV small (S)
segment RNA (T7 transcription kit, Fermentas, Vilnius, Lithuania).

Quantitative RT-PCR revealed the highest numbers of virus genome
copies in lungs and kidneys: 1,881 and 1,136 per Lig of total RNA,
respectively. Copy numbers per Lig of total RNA in other tissues were
lower: 240 in the heart, 160 in the spleen, 50 in the liver, and 42 in
the brain. In agreement with these findings, complete PUUV genome
sequences (12,059 nt) were recovered from the lung and kidney and
partial sequences of different lengths from heart, liver, and brain
(Figure 1). Corresponding sequences recovered from different tissues
were identical, i.e., no tissue-specific mutations were observed.

To determine whether this fatal NE case was caused by an unusual or
rare genetic variant of PUUV, we searched for identical or closely
related genetic variants in bank voles trapped near the patient's
house (storage buildings and surroundings within 500 m) in PieksAmAki,
central Finland (62[degrees]18'N, 27[degrees]08'E). Travel
history of the casepatient suggested that the infection had been
acquired at his residence. In 2008, the vole population peaked in the
southern half of Finland, including PieksAmAki, and 3,259 NE cases were
diagnosed nationwide (7), the highest number ever registered in Finland.
Sixty-three bank voles were snap-trapped during 3 consequent nights in
December 2008.

Lung tissue samples from the bank voles were screened for PUUV N
protein antigen by using immunoblotting, and 45 (71%) voles tested
positive. Tissues from 25 virusinfected voles were taken for genetic
analysis, and partial sequences of PUUV genome S, medium (M), and large
(L) segments (-12% of the total virus genome) were recovered from them.

[FIGURE 1 OMITTED]

In agreement with previously published data (10), the number of
PUUV genome copies in bank vole tissues was within the range of
105-106/Lig of total RNA, i.e., [approximately equals] 100-fold higher
than in tissues of the case-patient. Partial virus sequences from 4
voles were 100% identical to those from human tissues. Next, complete
PUUV genome sequences were recovered from 2 of these voles; the
sequences differed at only 4 positions in the L segment (all silent
mutations; Figure 1, right column). One of the complete
rodent-originated PUUV sequences was 100% identical to the sequence from
the case-patient. PUUV sequences have been deposited in GenBank under
accession nos. JN831943-JN831952. Phylogenetic analysis confirmed that
the hantavirus involved belonged to Puumala virus species and was most
closely related to the earlier described genetic variants from Finland,
particularly to those circulating at Konnevesi (62[degrees]34'N,
26[degrees]24'E) and Puumala (61[degrees]52'N,
28[degrees]17'E) localities (Figure 2).

Conclusions

Our findings established an unequivocal genetic link between the
fatal human NE case and local wild-type PUUV strains. These findings
also revealed that no mutations had accumulated in the genome of PUUV
during transmission of the virus to the patient and the fatal
generalized infection that followed. Finally, we demonstrated that the
wild-type PUUV strain that caused the fatal infection was neither a
unique nor rare genetic variant; the exact sequence match to the
complete human-originated PUUV sequence was found among the first 25
bank voles analyzed. Genetic links of the type have been reported for
PUUV infections in Finland (11) and for Sin Nombre virus infection
during the outbreak in the Four Corners area of the United States
(4,12), but perfect sequence match was not observed.

In PUUV infections, renal insufficiency is a hallmark of the
disease, but pulmonary, cardiac, central nervous system, ocular, and
hepatic manifestations and, in severe cases, hypophyseal injury, also
can occur (13). In the fatal case described here, death resulted from
multiorgan failure when kidneys, lungs, heart, and liver were affected.
The viral load was higher in the lungs and kidneys and lower in the
heart, spleen, liver, and brain. Whether this load distribution is
unique for fatal PUUV infections remains to be seen because
corresponding data for other hantavirus infections are missing.
Moreover, severe histopathologic changes were detected not only in lungs
and heart but also in liver and hypophysis, whereas kidneys, in this
respect, were almost normal. Thus, viral load does not seem to correlate
with tissue pathology. A more detailed pathologic description of this
and other lethal cases is under way.

[FIGURE 2 OMITTED]

Two more observations might be relevant to the case. First, human
leukocyte antigen typing showed that the patient had the risk haplotype
for severe NE, including a C4A null allele, i.e., a major antivirus
defense system complement was impaired (T. Sironen et al., unpub. data).
Second, the patient was a smoker and thus more likely to become infected
with PUUV (14). These factors might have substantially affected the
fatal outcome. We anticipate that our investigation will prompt further
full-length genome analyses of the wild-type strains of bunyaviruses
that cause infections in humans.

This work was supported by grants from The Academy of Finland,
Sigrid Juselius Foundation (Finland), and European Union grant
FP7-261504EDENext. This article is catalogued by the EDENext Steering
Committee as EDENext006 (www. edenext.eu).

Dr Plyusnina is an expert working with the Research Program Unit,
University of Helsinki. Her research interests include genetics and
evolution of hantaviruses.

(6.) Nemirov K, Lundkvist A, Vaheri A, Plyusnin A. Adaptation of
Puumala hantavirus to cell culture is associated with point mutations in
the coding region of the L segment and in the non-coding regions of the
S segment. J Virol. 2003;77:8793-800. http://dx.doi.
org/10.1128/JVI.77.16.8793-8800.2003